Kraft
mill effluent, due to its organic matter content and acute toxicity,
must be treated. A primary treatment followed by a secondary
treatment is the most common system. Aerated lagoon is also considered
an effective biological treatment, although this technology has some drawbacks
related with operation parameters and land extension space. Moreover,
the recovery efficiency for micropollutants contained in kraft
mill effluent is questioned due to the anoxic zone in the system.
The goal of this work is to evaluate the performance of the aerated
lagoon to remove stigmasterol contained in kraft mill effluents.
Kraft mill effluent was treated by an aerated lagoon (AL), which was operated
with three different stigmasterol load rates (SLR = 0.2, 0.6 and 1.1 mg/L x d)
and a hydraulic retention time of 1 day. The AL’s
maximum Chemical Oxygen Demand (COD) removal was 65%, whereas the Biological
Oxygen Demand (BOD5) was around 95%. The removal efficiency of
stigmasterol removal was 96% when SLR 1.1 mg/L x d, although an accumulation
of stigmasterol was detected for lower SLR.

Article

Estrogenic endocrine disruption could be
produced by extractive compounds coming from the kraft pulp. High
concentrations of extractive compounds (and in particular sterols) are
contained in black liquor from the digestion processes, and these are generally
recovered by evaporation and combustion (Xavier et al. 2005). However, there is
a small part of black liquor that remains in the fiber and is dragged into the
washing processes. Thus, in the discharged kraft mill effluents, concentrations
ranging from 0.3 to 3.4 mg/L for sterols and 0.28 to 1.21 mg/L for resin acids can
be found (Vidal et al. 2007). Moreover, physical chemical propiertes of stigmasterol
likes log Kow (10.20), molecular weight (412.7 g/gmol) or boiling
point (140ºC) show the persistant characteristics on the environment. Due to
above, recent studies have demonstrated the biological effects of these
compounds on fish in surface water systems (Orrego et al. 2006). The main
biological effects are alteration in the sexual steroid level (profile) in fish
plasma and diminished reproductive adaptation, among others (Larsson et al.
2002).

The most commonly used biological aerobic treatments
in kraft mills are aerated lagoons (Correa et al. 2003; Belmonte et al. 2006a)
and activated sludge (Khan and Hall, 2003). The main characteristics, of both systems
are the hydraulic conditions, biomass concentration and oxygen availability. Activated
sludge and aerated lagoons are easy to operate but require large hydraulic
retention time (HRT) and elevated land extensions. Biodegradable organic matter
(85-95%) and acute toxicity (100%) could be removed (Kostamo and Kukkonen, 2003;
Belmonte et al. 2006b). However, the operating conditions strongly influence
the degradation of endocrine disrupting chemicals (EDC) (Werker
and Hall, 1999). Vidal et al. 2007 demonstrated that in anoxic areas, intermediate
compounds of the resin acid biodegradation can produce accumulation. Moreover, Kostamo
and Kukkonen (2003) have shown that over 41% of the sterols were reduced or
transformed into other compounds. Furthermore, in general, less than 5% of the
resin acids and over 31% of the sterols were removed in biosludge of the sludge
thickener. On the other hand, Khan and Hall (2003) and Kostamo et al. (2004) have obtained percentages of phytosterol adsorption from 30 to 70 % in biomass
of AS and AL systems.

Cook et al. (1997) worked with 5 kraft
cellulose plants where the whitening process consisted in elementary chlorine
free processes, finding that a phytosterol reduction process was produced in
all cases except in the case of stigmasterol, where it could increase between
192% and 367%. In the activated sludge system, an 88% increase in stigmasterol
was observed, and is probably due to the chemical or biological transformation
in the precursor treatment system where it was converted into stigmasterol (Cook
et al. 1997). However, few studies present results with respect to EDC behavior
in aerated lagoons.

The
goal of this work is to evaluate the performance of aerated lagoon in removing stigmasterol
contained in kraft mill effluents.

Materials
and Methods

Wastewater

The effluent was obtained from a modern
kraft mill that bleaches softwood pulp and is located in Southern Chile. Pinus
radiata is the raw material used in the process. The kraft mill effluent
was obtained after primary treatment. Table 1 shows the main physicochemical
characteristics of this effluent. The effluent was supplemented with 0.022 g/L of
NH4Cl as nitrogen source and 0.169 g/L of K2PO4 as phosphorus source (BOD5:N:P = 100:5:1).

Inoculum

The aerobic lagoon was inoculated using an
aerobic microbial consortium (5 g/L of volatile suspended solid (VSS), 9 g/L of
total suspended solid (TSS)) that was obtained from an aerobic sewage treatment
plant without a nitrification/denitrification process.

Continuous bioreactor system

An
aerated lagoon (AL) with an aerated area (0.44 L) and a settling
(non-aerated) area (0.22 L) was used as biological treatment, following Correa
et al. (2003). A
peristaltic pump fed the system. The oxygen concentration in the
aerated zone was maintained above 6 mg/L using a diffuser system.
The AL operation was divided in two phases. In Phase I, the system
was fed with only kraft mill effluent to establish steady-state
performance conditions. The HRT of the system was maintained for
24.0 hrs, corresponding to organic load rates (OLR) around 0.81
g COD/L x d. In Phase II, the lagoon was fed with kraft mill effluent
supplemented with an increasing concentration of stigmasterol (Merck,
95%) (concentration ranged from 0.2 to 1 mg/L). The stigmasterol
was dispersed into kraft mill effluent matrix by sonication during
10 hrs. The HRT was reduced stepwise from 24.0 to 12.4 hrs, resulting
in an increase of the Stigmasterol Load Rate (SLR) from 0.55 to
1.1 mg/L d. The removal efficiencies of BOD5, COD, total
phenolic compounds, stigmasterol and color levels were calculated
using equation 1. Solids were not removed from the reactor.

VSS, TSS, COD, BOD5, phenols,
and lignin and tannins were measured following Standard Methods
(APHA-AWWA-WPCF, 1985). The total phenolic compounds (UV phenols) concentration
was measured by UV absorbance in a 1-cm quartz cell at 215 nm, pH 8.0 (0.2 M KH2PO4 buffer) and transformed to concentration using a calibration curve
with phenol as standard solution. The samples for parameter determination were
membrane filtered (0.45 µm). Spectrophotometric measurements of filtered
samples were principally performed at wavelengths of 436 (color), 346
(lignosulfonic acids), 254 (aromatic compounds) and 280 (lignin derived
compounds) in a 1 x 1-cm quartz cell using a Genesys UV-VIS spectrophotometer,
and were determined according to the Çeçen (2003) procedure.

The
stigmasterol present in the kraft mill effluent was first identified
in samples filtered though a 0.2-µm membrane
filter by gas chromatography CG-MS (HP 5890 chromatograph with mass
detector HP 5972) using a column Agilent (19091s-433 HP-MS 5% phenyl-methyl-siloxane,
length 30 m, internal diameter 0.5 µm). The detection limit
was fixed at 1 µg/L. The
compounds were extracted from 100 mL of samples with 20 mL dichlormethane
at pH value of 7. The extract was rota-evaporated and re-suspended
in 2 mL of chloroform and injected to the chromatograph (Cook
et al. 1997;
Khan and Hall, 2003). Patron solutions were prepared
with stigmasterol (Sigma) using cholesterol (Carbiochem) as internal
patron and a calibration curve prepared at concentrations from 4
to 130 mg/L. Samples were extracted following the procedure used
for the previous qualitative analysis and spiked with 1000 mg/L of
cholesterol. A volume of 1 µL was injected to the GC-FID
chromatograph. Stigmasterol values were determined in 50 mL of sludge
samples filtered and dried (105ºC for 12 hrs). The dried sample
was extracted in a Soxhlet with 200 mL of dichloromethane for 20
hrs. After this point, the same procedure used for the quantitative
determination was used.

The acute toxicity of influent and
effluent on D. magna (< 24 hrs old) was evaluated at 24 hrs.
Mortality was recorded at the end of exposure, where mortality was defined as a
lack of organism mobility when the vessel was shaken. Five samples with
different concentrations (6.25, 12.5, 25, 50, 100%) and one control were
evaluated. Four replicates of 30 mL (each one containing five organisms) were
performed for each concentration and the control. The culture was not renewed
during the test. Oxygen concentration, pH and conductivity were measured at the
beginning and end of each test. The 24 hrs mean lethal concentrations were
calculated using the Probit and the Spearman-Karber methods, as appropriate (USEPA,
1993; NCh 2083, 1999).

Results and Discussion

Figure
1 shows the performance of the AL. HRT was maintained around
1 day to evaluate the behavior of stigmasterol biodegradation
and the biomass evolution. During the operation of Phase I, the
OLR was on average maintained around 0.81 g COD/L x d; whereas
during the second phase, the OLR increased until 0.96 g COD/L x
d. On the other hand, the average SLR values during Phase II
were 0.2, 0.6 and 1.1 mg/L x d, respectively.

In spite of this difference, it was
observed that total phenolic compounds were removed by the AL, and that the
color and aromatic compounds were polymetizated in the aerobic system. Due to
this, Figure 1c shows negatives removal values. These phenomena were observed
in previous work. In fact, Chamorro et al. (2005) show that aromatic compounds of
the higher molecular weight fraction (from 5.000 to 10.000 Da) increased after an
AL biological treatment. This phenomenon is due to the oxidation-polymerization
process (Milestone et al. 2004).

Table 2 shows the behavior of aromatic compounds with respect
to COD during the kraft mill effluent treatment by AL assays. Most of these
parameters show the degradation and non-biodegradable fraction in the kraft
mill effluent (Çeçen, 1999). The behavior of lignosulfonic acid (VIS346/COD)
content in the kraft mill effluent is in a range of 0.283 - 0.448. The
relationship VIS346/COD increased, indicating low biodegradation of
these compounds by aerobic bacteria. In the same way, lignin behavior (measured
as UV280/COD) shows a range between 0.026 - 0.051 in the different assays, indicating that aerobic biomass mineralizes the lignin compounds to a
lesser extent than organic matter (measured as COD).

The UV254/UV280 relationship
is used as an indicator of lignin-derived compound presence in wastewaters,
where low values indicate a higher percentage of these compounds (Çeçen, 2003).
During the AL treatment process, the UV254/UV280 relationship
in the effluent was around 1.26 - 1.28. Similarly, Çeçen (2003) shows that UV254/UV280 did not undergo a significant change (ranging between 1.1 - 1.13). These
results suggest that the residual COD consisted in lignin compounds, which were
also the major aromatic species in these effluents. The same results were found
by Chamorro et al. (2005) and Milestone et al. (2004).

VIS400 and VIS436 show color behavior during the aerobic treatment. Low values show that
recalcitrant compounds are present in the treated effluent (Çeçen, 1999; Çeçen,
2003). Color behavior in the system is also explained by the no biodegradation
of lignin and aromatic compounds in general.

Figure
2 shows the stigmasterol removal in the AL
system. Stigmasterol increases at low SLR, ranging from 29 - 37%.
This phenomenon was also observed by Cook et al. (1997),
who found that stigmasterol could increase its concentration in more
than 300% in AL system. Also, Khan and Hall
(2003) show that aerated systems are not efficient for stigmasterol
removal. However, when SLR increases up to 0.6 mg/L x d, the removal
efficiency of stigmasterol increased to 90%. The sterol biodegradation
mechanisms are biotransformation and adsorption (Fernandez
et al. 2007). Khan and Hall (2003) and Kostamo
and Kukkonen (2003) have obtained percentages of phytosterol
adsorption from 30 to 70% in AL system biomass.

Bioassays
with D. magna indicate
that AL treatment of kraft mill effluent can only partially remove
the acute toxic compounds (toxicity reduction range between 23.12%
and 40.64%) at low SLR (0.2 mg/L x d) (Figure
3). However, when SLR
increases up 0.6 mg/L x d, the 24 hrs LC50 values of
the aerobic effluent are above 100%. A similar effect was found by Priha
(1996) in the evaluation of 13 industries with activated sludge
and AL.

Concluding Remarks

Organic matter removal by AL ranged
between 25.0 - 65.2% for COD, BOD5 removal was above 95%, and total
phenolic compounds removal was around 48%.

Under
experimental conditions, stigmasterol removal was 96% when SRL
increases up to 0.6 mg/L x d. Stigmasterol removal was strongly
dependent on the operation rate. Stigmasterol fate needs to be
studied in the future.

Acknowledgments

This work was supported by the FONDECYT
Grant 1070509 and Ph. D. Thesis support fellowship (First year). FONDECYT for
International-Cooperation Support, Grant 7080172.